Labeling and anti-drug antibody assays for AAV vectors

Abstract

The present invention relates, in general terms, to a method for testing for the presence of anti-drug antibodies (ADAs) against therapeutic viral vectors. In particular, the present invention relates to the use of biotin-labeled AAV as a capture reagent and ruthenium-labeled AAV as a detection reagent for the detection and quantification of ADAs against AAVs.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 464,277, filed May 5, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Adeno - associated virus (AAV) is widely used as a gene delivery vector for delivering genetic materials such as the delivery of nucleic acids for gene therapy. As a whole, AAV - based therapies have the advantages of being non - pathogenic and non - toxic, having cell - type - specific infection, and providing different serotypes with various transduction efficiencies. The drawback is that the production, purification, and characterization of AAV are more complex compared to, for example, antibody therapies.

[0003] Wild - type AAV is clearly a non - pathogenic virus, but infection with wild - type AAV is common, and 30 - 60% of all individuals may carry existing antibodies that can neutralize AAV transduction. Therefore, assays for detecting and quantifying existing antibodies should be included in the evaluation of clinical immunogenicity. An efficient method for characterizing AAV anti - drug antibodies (ADA) comparable to the ADA assay for antibody therapeutics does not yet exist. The ADA assay typically uses a labeled drug as both a capture reagent and a detection reagent and uses a bridging format. For example, ADA can bind to both a biotin - labeled drug and a ruthenium - labeled drug and form a bridge between the two. The bridged complex is captured by the streptavidin - coated plate surface. Current is used to activate the ruthenium - labeled drug to generate an electrochemiluminescence signal, which can be used to detect the presence of ADA.

[0004] The relatively small number of accessible (surface-exposed) lysine atoms compared to the size of the AAV presents inherent challenges for lysine-directed conjugation of labels and for assays that depend on labeled analytes, such as ADA crosslinking assays. Furthermore, the large size of the AAV makes conventional methods for determining labeling efficiency, such as matrix-assisted laser desorption / ionization (MALDI), unsuitable.

[0005] Therefore, it will be understood that there is a need for methods and compositions for labeling AAV vectors, determining the degree of labeling of AAV vectors, and detecting anti-drug antibodies against AAV vectors. [Overview of the project]

[0006] overview This disclosure provides a method for detecting and / or quantifying antibodies against a viral capsid of interest in a sample. In some exemplary embodiments, the method may include (a) contacting a sample with a capture reagent, a detection reagent, and a solid surface to form a detection mixture such that the capture reagent and the detection reagent can bind to antibodies against the viral capsid of interest, and the capture reagent can bind to the solid surface; and (b) measuring the detection reagent to detect and / or quantify antibodies against the viral capsid of interest in the sample.

[0007] In one embodiment, the sample is serum. In a particular embodiment, the serum is human serum.

[0008] In one embodiment, the capture reagent contains biotin.

[0009] In one embodiment, the capture reagent includes the viral capsid of interest.

[0010] In one embodiment, the detection reagent includes radiolabeling, phosphorescent labeling, chemiluminescence labeling, fluorescent labeling, fluorophores, haptens, electrochemiluminescence labeling, or enzyme labeling. In a particular embodiment, the detection reagent includes ruthenium or horseradish peroxidase.

[0011] In one embodiment, the detection reagent comprises an antibody. In another embodiment, the detection reagent comprises the viral capsid of interest.

[0012] In one embodiment, the solid surface is selected from the group consisting of microplates, resins, agarose beads, and magnetic beads. In another embodiment, the solid surface is coated with avidin or streptavidin.

[0013] In one embodiment, the antibody against the viral capsid of interest is an anti-drug antibody.

[0014] In one embodiment, the viral capsid of interest is an AAV capsid. In a particular embodiment, the serotype of the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, combinations thereof, or variants thereof. In another particular embodiment, the AAV is a viral vector. In a more particular embodiment, the AAV is a therapeutic viral vector.

[0015] In one embodiment, the viral capsid is a non-enveloped viral capsid.

[0016] The disclosure also provides a method for detecting anti-drug antibodies against an AAV vector. In some exemplary embodiments, the method may include (a) contacting serum with a capture reagent, a detection reagent, and a solid surface coated with avidin or streptavidin to form a detection mixture; (b) subjecting the detection mixture to an electric current to generate a signal; and (c) measuring the signal to detect anti-drug antibodies against an AAV vector, wherein the capture reagent comprises the AAV vector conjugated to biotin, and the detection reagent comprises the AAV vector conjugated to ruthenium.

[0017] In one embodiment, the serum is human serum.

[0018] In one embodiment, the solid surface is selected from the group consisting of microplates, resin, agarose beads, and magnetic beads.

[0019] In one embodiment, the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

[0020] In one embodiment, the AAV vector is a therapeutic vector.

[0021] In one embodiment, the signal is an electrochemiluminescence signal.

[0022] The Disclosure further provides compositions for detecting and / or quantifying antibodies against a viral capsid of interest in a sample. In some exemplary embodiments, the composition may comprise (a) a sample, (b) a capture reagent, (c) a detection reagent, and (d) a solid surface, wherein the capture reagent and the detection reagent can bind to antibodies against the viral capsid of interest, and the capture reagent can bind to the solid surface.

[0023] In one aspect, the sample is serum. In certain aspects, the serum is human serum.

[0024] In one aspect, the capture reagent includes biotin.

[0025] In one aspect, the capture reagent includes the viral capsid of interest.

[0026] In one aspect, the detection reagent includes a radiolabel, a phosphorescent label, a chemiluminescent label, a fluorescent label, a fluorophore, a hapten, an electrochemiluminescent label, or an enzyme label. In certain aspects, the detection reagent includes ruthenium or horseradish peroxidase.

[0027] In one aspect, the detection reagent includes an antibody. In another aspect, the detection reagent includes the viral capsid of interest.

[0028] In one aspect, the solid surface is selected from the group consisting of microplates, resins, agarose beads, and magnetic beads. In another aspect, the solid surface is coated with avidin or streptavidin.

[0029] In one aspect, the antibody against the viral capsid of interest is a drug - resistant antibody.

[0030] In one aspect, the viral capsid of interest is an AAV capsid. In certain aspects, the serotype of the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, combinations thereof, or variants thereof.

[0031] In one aspect, the AAV is a viral vector. In certain aspects, the AAV is a therapeutic viral vector.

[0032] In one embodiment, the viral capsid is a non-enveloped viral capsid.

[0033] This disclosure further provides compositions for detecting anti-drug antibodies against AAV vectors. In some exemplary embodiments, the composition may comprise (a) a sample, (b) a biotin-conjugated AAV vector, (c) a ruthenium-conjugated AAV vector, and (d) a solid surface coated with avidin or streptavidin, wherein the biotin-conjugated AAV vector and the ruthenium-conjugated AAV vector are modified versions of the same AAV vector.

[0034] In one embodiment, the sample is serum. In a particular embodiment, the serum is human serum.

[0035] In one embodiment, the solid surface is selected from the group consisting of microplates, resin, agarose beads, and magnetic beads.

[0036] In one embodiment, the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

[0037] In one embodiment, the AAV vector is a therapeutic vector.

[0038] The disclosure also provides a method for generating a labeled viral vector. In some exemplary embodiments, the method may include (a) buffer-exchange a sample containing a viral vector to generate a viral vector sample having a basic pH; (b) contact the viral vector sample having a basic pH with a label-linked N-hydroxysuccinimide ester to generate a mixed sample containing a labeled viral vector; and (c) purify the mixed sample to generate a labeled viral vector.

[0039] In one embodiment, the labeled reagent was purified three times using a Zeba Spin Desalting column 40K MWCO to remove any unlabeled dyes.

[0040] In one embodiment, the label is selected from the group consisting of radioactive labels, phosphorescent labels, chemiluminescent labels, fluorescent labels, fluorophores, haptens, affinity labels, electrochemiluminescent labels, and enzyme labels. In a particular embodiment, the label is selected from the group consisting of biotin, ruthenium, and horseradish peroxidase.

[0041] In one embodiment, the viral vector is an AAV vector. In a particular embodiment, the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, combinations thereof, or variants thereof.

[0042] In one embodiment, the concentration of the viral vector in the viral vector sample having a basic pH is approximately 10 per 1 mL. 12 ~about 10 13 It is a vector genome.

[0043] In one embodiment, the molar ratio of the N-hydroxysuccinimide ester linked to the label to the viral vector is approximately 50:1 to approximately 20,000:1. In a particular embodiment, the molar ratio is approximately 10,000:1.

[0044] In one embodiment, the viral vector is a non-enveloped viral vector.

[0045] In one embodiment, the purification includes replacing the buffer in the mixed sample to remove any unconjugated labels.

[0046] Additional information provided in this disclosure provides a method for determining the degree of labeling of a viral capsid. In some exemplary embodiments, the method may include (a) determining the concentration of a conjugated label in a labeled viral capsid sample; (b) determining the concentration of a viral capsid in the labeled viral capsid sample; and (c) determining the degree of labeling of the viral capsid by dividing the concentration in (a) by the concentration in (b).

[0047] In one embodiment, the viral capsid is an AAV capsid. In a particular embodiment, the serotype of the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, combinations thereof, or variants thereof.

[0048] In one embodiment, the label is selected from the group consisting of radioactive labels, phosphorescent labels, chemiluminescent labels, fluorescent labels, fluorophores, haptens, affinity labels, electrochemiluminescent labels, and enzyme labels. In a particular embodiment, the label is ruthenium.

[0049] In one embodiment, determining the concentration of a conjugated label includes (a) subjecting the labeled viral capsid sample to size exclusion chromatography; (b) measuring the light absorption at a wavelength corresponding to the absorption maximum of the label; (c) integrating the area under the curve corresponding to the main peak of the labeled viral capsid at the wavelength of (b) to determine the peak area; and (d) comparing the peak area with a standard curve relating peak area to concentration to determine the concentration of the conjugated label.

[0050] In one embodiment, the wavelength is approximately 450 nm.

[0051] These and other aspects of the present invention will be better recognized and understood when considered in conjunction with the following description and accompanying drawings. The following description illustrates various embodiments and numerous specific details thereof, but these are given as examples only and are not limiting. Many substitutions, modifications, additions, or reconfigurations can be made within the scope of the present invention. [Brief explanation of the drawing]

[0052] [Figure 1] Figure 1A shows a diagram of the monomeric structure of the AAV VP protein according to an exemplary embodiment, where VP1 is the largest monomer. Figure 1B shows a comparison of different AAV serotypes and their identity percentages according to an exemplary embodiment. [Figure 2] Figure 2 illustrates several analytical methods for feature analysis and monitoring of the unique quality attributes of AAVs, according to exemplary embodiments. [Figure 3] Figure 3 shows a schematic diagram of a conventional cross-linked anti-drug antibody (ADA) assay format for a monoclonal antibody according to an exemplary embodiment. [Figure 4] Figure 4A shows a schematic diagram of an ADA assay using biotin-labeled AAV as the capture reagent and HRP-labeled anti-mouse IgG as the detection reagent, according to an exemplary embodiment. Figure 4B shows a schematic diagram of an ADA assay using biotin-labeled AAV as the capture reagent and ruthenium-labeled AAV as the detection reagent, according to an exemplary embodiment. [Figure 5] Figure 5A shows the stability of AAV1 in buffers of various pH values, as measured by the polydispersity index (PDI), according to an exemplary embodiment. Figure 5B shows the stability of AAV8 in buffers of various pH values, as measured by the polydispersity index (PDI), according to an exemplary embodiment. [Figure 6A] Figure 6A shows an exemplary method for labeling AAV, comprising buffer exchange to basic pH, a challenge ratio of 100 to 10000x, and purification of the labeled AAV, according to an exemplary embodiment. [Figure 6B]Figure 6B shows a size exclusion chromatography (SEC) chromatogram of biotin-labeled and purified AAV according to an exemplary embodiment, where biotin-labeled AAV was measured at a retention time of approximately 12.50 minutes, and excess biotin was measured at a retention time of approximately 18.70 minutes. [Figure 7A] Figure 7A shows a schematic diagram of an ADA assay using a mouse anti-AAV8 mAb and anti-mouse IgG-HRP (detection reagent) and biotin-labeled AAV8 (capture reagent) that forms crosslinking molecules, according to an exemplary embodiment. [Figure 7B] Figure 7B shows the effect of biotin-labeled AAV concentration on the detected signal using both monoclonal and polyclonal antibodies derived from rabbits and mice, according to exemplary embodiments. [Figure 7C] Figure 7C shows the signals generated from an ADA assay using normal human serum and biotin-labeled AAV8 as capture reagents, according to an exemplary embodiment. [Figure 8] Figure 8 shows the SEC chromatograms of successfully ruthenium-labeled and purified AAV, measured at a retention time of approximately 12.50 minutes and with excess ruthenium measured at a retention time of approximately 18.70 minutes, according to an exemplary embodiment. [Figure 9A] Figure 9A shows an exemplary method for calculating the degree of labeling of a labeled AAV according to an exemplary embodiment. [Figure 9B] Figure 9B shows the range of ruthenium peak areas, measured at absorbance of 450 nm, corresponding to a range of ruthenium concentrations according to an exemplary embodiment. [Figure 9C] Figure 9C shows a standard curve relating the peak area of ​​ruthenium, measured at absorbance at 450 nm, to the concentration of ruthenium in μM, according to an exemplary embodiment. [Figure 9D] Figure 9D shows the calculated degree of labeling (DOL) of a ruthenium-labeled monoclonal antibody compared to ruthenium-labeled AAV at two different challenge ratios (1,000:1 and 10,000:1) according to an exemplary embodiment. [Figure 10A] Figure 10A shows the amount of excess ruthenium remaining after ruthenium conjugation with AAV and various buffer exchange conditions according to an exemplary embodiment. [Figure 10B] Figure 10B shows the AAV and excess ruthenium recovered after ruthenium conjugation with AAV and various buffer exchange conditions, according to an exemplary embodiment. [Figure 11] Figure 11A shows signals from an ADA crosslinking assay using biotin-labeled AAV8 as the capture reagent and ruthenium-labeled AAV8 as the detection reagent, according to an exemplary embodiment, at two different challenge ratios. Figure 11B shows signals from an ADA crosslinking assay using biotin-labeled AAV8 as the capture reagent and ruthenium-labeled AAV8 as the detection reagent, according to an exemplary embodiment, at various AAV8 concentrations and a 10,000:1 challenge ratio. [Figure 12] Figure 12 shows signals from an ADA crosslinking assay using biotin-labeled AAV8 as the capture reagent and ruthenium-labeled AAV8 as the detection reagent for normal human serum, according to an exemplary embodiment. [Modes for carrying out the invention]

[0053] Detailed explanation Adeno-associated viruses (AAVs) are widely used as gene delivery vectors for delivering genetic material, such as for the delivery of nucleic acids for gene therapy. AAVs offer the advantages of being non-pathogenic and having low immunogenicity. AAVs are non-pathogenic members of the Parvoviridae family under the genus Dependovirus and require a helper virus, such as an adenovirus or herpesvirus, for infection (Venkatakrishnan et al., Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1- Unique N-Terminal Domain and Its Role in Capsid Trafficking, Journal of Virology, May, 2013, vol. 87, no. 9, pages 4974-4984). AAVs encapsulate a single-stranded DNA genome of approximately 4.8 kilobases (kb) in an icosahedral capsid constructed from a shell of capsid virus protein. Recombinant AAV genomes are non-pathogenic and do not integrate into the host genome, but exist as stable episomes that provide long-term expression. AAV serotypes are a very useful system for preferentially transducing specific cell types.

[0054] Overall, AAV-based therapies have the advantages of being non-pathogenic and non-toxic, possessing cell-type specific infection, and offering different serotypes with varying cytotransduction efficiencies. The disadvantage is that AAV production, purification, and characterization are more complex compared to, for example, antibody therapies. Fully packaged AAV consists of an icosahedral capsid containing a single-stranded genome of approximately 4.7 kb. An empty capsid has a molecular weight of approximately 3750 kDa, while a full capsid with a single-stranded genome of approximately 4.7 kb has a molecular weight of approximately 5100 kDa. The purity of AAV is defined by several product-related impurities, including empty capsids, capsids containing partial or inaccurate genomes, and aggregated or degraded capsids.

[0055] The AAV capsid is composed of 60 subunits assembled from three proteins: VP1, VP2, and VP3, in a ratio of approximately 1:1:10. These VP proteins share a common C-terminus, as shown in Figure 1A. VP monomers form both trimers and pentamers, which associate to form the complete capsid. The core capsid structure is shared across different serotypes. The greatest sequence variation is observed in the surface-exposed loop. A comparison of sequence identity between serotypes is shown in Figure 1B.

[0056] Wild-type AAV is clearly a non-pathogenic virus, but infection with wild-type AAV is common, and 30–60% of all individuals may possess pre-existing antibodies that can neutralize AAV transduction. Therefore, assays to detect and quantify pre-existing antibodies should be included in the assessment of clinical immunogenicity. Immunogenicity of pharmaceuticals, including viral vectors such as AAV vectors, is a major concern in clinical and preclinical studies because it can lead to potentially serious side effects, loss of efficacy, and altered drug exposure, complicating the interpretation of toxicity, pharmacokinetic (PK), and pharmacodynamic (PD) data.

[0057] As shown in Figure 2, various analytical techniques have been developed to characterize and monitor the intrinsic quality attributes of AAVs, including isoform purity, ITR deletion, size heterogeneity, charge heterogeneity, titer, thermal stability, and capsid empty / full ratio. However, an efficient method for characterizing AAV anti-drug antibodies (ADAs) comparable to ADA assays for antibody therapeutics still does not exist. ADA immunoassays for detecting and quantifying ADAs are crucial in determining the immunogenicity of biological therapies. ADA assays typically use a cross-linked form that uses the drug as both the capture and detection reagent. These assays are relatively easy to set up and run, detect most isotype responses, and offer excellent sensitivity. They are species-nonspecific and high-throughput.

[0058] A conventional ADA format for monoclonal antibodies commonly used throughout the industry is shown in Figure 3. Any ADA binds to both a biotin-labeled antibody drug and a ruthenium-labeled antibody drug, forming a crosslink between the two. The crosslinked complex is captured by the streptavidin-coated plate surface. An electric current activates the ruthenium-labeled drug to produce an electrochemiluminescent signal, which can be used to detect the presence of ADA. The assay is highly sensitive, detects all ADA isotypes, and is useful for non-quantitative titer-based assessment of immunogenicity. Additional potential ADA assay formats using examples of AAV drugs are shown in Figures 4A and 4B.

[0059] Table 1 shows a comparison of AAVs and antibodies for the purpose of characteristic analysis and labeling conjugates. The relatively small number of accessible (surface-exposed) lysines compared to the size of the AAV presents inherent challenges for lysine-directed conjugation of labeling and for assays that depend on the labeled analyte, such as the ADA crosslinking assay. Furthermore, due to the large size of the AAV, conventional methods for determining labeling efficiency, such as matrix-assisted laser desorption / ionization (MALDI), are not applicable.

[0060] (Table 1) Physical properties of antibodies and AAV TIFF2026516022000002.tif31128

[0061] As described above, there is a need for methods and compositions for labeling AAV vectors for detection and for detecting and quantifying anti-drug antibodies against therapeutic AAVs. This disclosure describes methods and compositions for labeling the capsid of an AAV vector, determining the degree of AAV labeling, and using the labeled AAV or AAV capsid to detect and quantify anti-drug antibodies against an AAV vector.

[0062] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Any methods and materials similar to or equivalent to those described herein may be used for testing, but specific methods and materials are described herein.

[0063] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation, as understood by those skilled in the art, and to include endpoints where a range is provided. As used herein, the terms "include," "includes," and "including" are intended to be non-limiting and should be understood to mean "comprise," "comprises," and "comprising," respectively.

[0064] As used herein, the terms “protein” or “protein of interest” may include any amino acid polymer having covalent amide bonds. A protein comprises one or more amino acid polymer chains commonly known in the art as a “polypeptide.” A “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and non-naturally occurring analogs of its synthesis, linked via peptide bonds. A “synthetic peptide or polypeptide” refers to a peptide or polypeptide that does not exist in nature. Synthetic peptides or polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein may comprise one or more polypeptides to form a single functional biomolecule. A protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. The proteins of interest may include any of the following: biological therapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins may be produced using recombinant cell line production systems, such as insect baculovirus lines, yeast lines (e.g., Pichia), and mammalian lines (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation” (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012)) (their entire teachings are incorporated herein by reference). Proteins can be classified based on their composition and solubility, and thus may include simple proteins such as globular and fibrous proteins, complex proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins, as well as inducible proteins such as primary and secondary inducible proteins.

[0065] As used herein, the term “recombinant protein” refers to a protein produced as a result of the transcription and translation of a gene supported on a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an isotype antibody selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule may be a full-length antibody (e.g., IgG1 or IgG4 immunoglobulin), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0066] As used herein, the term “antibody” includes an immunoglobulin molecule comprising four polypeptide chains, namely two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and its polymer (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions may be further subdivided into a hypervariable region called the complementarity-determining region (CDR), which contains interspersed more conserved regions called the framework region (FR). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs of the anti-big-ET-1 antibody (or its antigen-binding moiety) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms “antigen-binding moiety” of an antibody, “antigen-binding fragment” of an antibody, and similar include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody may be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding the antibody variable domain and optionally a constant domain. Such DNA is publicly known and / or may be readily available from, for example, commercial sources, DNA libraries (e.g., including phage antibody libraries), or can be synthesized.DNA may be sequenced and manipulated chemically or by using molecular biological techniques, for example, by arranging one or more variable domains and / or constant domains in a preferred configuration, introducing codons, generating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0067] As used herein, “antibody fragment” includes, for example, a portion of a complete antibody, such as the antigen-binding region or variable region of an antibody. Examples of antibody fragments, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolation complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of the immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the immunoglobulin light and heavy chain variable regions are linked by a peptide linker. In some exemplary embodiments, an antibody fragment comprises the full amino acid sequence of the parent antibody, and in some exemplary embodiments, the fragment binds to the antigen with an affinity equivalent to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments can be produced by any means. For example, antibody fragments may be produced enzymatically or chemically by fragmentation of a complete antibody, and / or recombinantly from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments may be produced entirely or partially synthetically. Antibody fragments may optionally include single-chain antibody fragments. Alternatively or additionally, antibody fragments may include multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, and more typically, at least about 200 amino acids.

[0068] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. A bispecific antibody generally contains two distinct heavy chains, each specifically binding to either two different molecules (e.g., multiple antigens) or the same molecule (e.g., the same antigen). If a bispecific antibody can selectively bind to two different epitopes (a first and a second epitope), the affinity of the first heavy chain to the first epitope is generally at least one to two orders of magnitude, or even three or four orders of magnitude, lower than the affinity of the first heavy chain to the second epitope, and vice versa. The epitopes recognized by a bispecific antibody may be on the same target or different targets (e.g., on the same protein or different proteins). A bispecific antibody can be constructed, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable regions that recognize different epitopes of the same antigen can be fused with nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains.

[0069] A typical bispecific antibody comprises two heavy chains, each having three heavy chain CDRs followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain; and an immunoglobulin light chain that does not confer antigen-binding specificity but is either associable with each heavy chain, or associable with each heavy chain and capable of binding one or more epitopes that bind to the heavy chain antigen-binding region, or associable with each heavy chain and capable of binding one or both of the heavy chains to one or both epitopes. BsAbs can be divided into two main classes: those possessing an Fc region (IgG-like) and those lacking an Fc region, the latter of which are usually smaller than IgG and IgG-like bispecific molecules that contain Fc. IgG-like bsAbs include, but are not limited to, triomab, knob-in-hole IgG (kih IgG), crossMab, orth-Fab IgG, dual-variable domain Ig (DVD-Ig), two-in-one or dual-acting Fab (DAF), single-stranded IgG Fv (IgG-scFv), or κλ bodies, and may have different forms. Non-IgG-like alternative formats include tandem scFv, diabody format, single-stranded diabody, tandem diabody (TandAb), biaffinity retargeting molecule (DART), DART-Fc, nanobody, or antibodies produced by the Dock-and-Lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the entire teachings of which are incorporated herein).

[0070] As used herein, “multispecific antibody” refers to an antibody that has binding specificity to at least two different antigens. Such molecules typically bind to only two antigens (i.e., bispecific antibody, bsAb), but antibodies with further specificity, such as triplicate antibodies and KIH triplicate, can also be addressed by the systems and methods disclosed herein.

[0071] As used herein, the term “monoclonal antibody” is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies may be derived from a single clone, including any eukaryotic cell, prokaryotic cell, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in this disclosure may be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombination, and phage display technologies, or combinations thereof.

[0072] In some exemplary embodiments, recombinant proteins may be produced from mammalian cells. Mammalian cells may be of human or non-human origin, and include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their lineages (e.g., 293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells, or their derivatives), fibroblasts from any tissue or organ (but not limited to, heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph glands, pharyngeal tonsils, tonsils, bone marrow, and blood), spleen), as well as fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, B LO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian This may include muntjac cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells, or derivatives thereof.

[0073] As used herein, the terms “anti-drug antibody” or “ADA” refer to antibodies produced by the immune system of interest that target an epitope on a therapeutic protein or viral vector. The term “drug” should be understood to include monomeric proteins, macrometic proteins, small molecules, viral vectors, or any other such chemical entities produced for therapeutic or diagnostic purposes. Anti-drug antibodies may develop during therapy as an immunogenic response in a patient. In the case of viral vector therapy, a patient may have ADAs due to prior exposure to the virus in question. It should be understood that ADAs are not limited to antibodies produced in response to viral vector therapy, but include antibodies that may arise in response to wild-type viruses of the same or similar viral or serotype as the viral vector of interest. For example, an antibody that may be produced in response to wild-type AAV8 infection would be considered an ADA in the context of the AAV8 viral vector, or any viral vector to which the antibody may be conjugated.

[0074] A subset of ADAs are “neutralizing antibodies” or “NAbs” that can bind to therapeutic proteins, viral vectors, or other drugs in a manner that inhibits or neutralizes their pharmacological activity, for example, by inhibiting transduction by viral vectors. NAbs may affect the clinical efficacy of therapeutic proteins or viral vectors and therefore must be monitored when administering them to therapeutic proteins.

[0075] ADA immunoassays are well known to those skilled in the art. Such assays, as well as practical applications and methods for carrying out the procedures, are well known in the art, for example, in Colowick, SP and Caplan, NO (eds.), “Methods in Enzymology”, Academic Press, dealing with immunological detection methods, particularly in volumes 70, 73, 74, 84, 92 and 121. The principles of different immunoassays are described, for example, in Hage, DS (Anal. Chem. 71 (1999) 294R-304R) and Lu, B., et al. (Analyst 121 (1996) 29R-32R), describing the orientation and immobilization of antibodies for use in immunoassays. For avidin-biotin-mediated immunoassays, see, for example, Wilchek, M., and Bayer, EA, in Methods Enzymol. 184 (1990) 467-469.

[0076] The commonly used ADA assay method is a cross-linked immunoassay (see, for example, Liao, K., et al., J Immunol Methods, 2017, 441: p. 15-23, Dai, S., et al., AAPS J, 2014, 16(3): p. 464-77, and Zhong, ZD, et al., AAPS J, 2017. 19(6): p. 1564-1575 (the contents of which are incorporated herein by reference)). The ADA cross-linked immunoassay is a sandwich-type immunoassay in which polyvalent ADA is conjugated by a capture reagent and a detection reagent, and optionally one or both of the reagents are the drug of interest, each conjugating to a different, non-overlapping, or non-interfering epitope of ADA. The capture reagent and / or detection reagent may be the drug of interest, or a therapeutic protein, or a vector, or an antibody targeting ADA. In particular, in this assay, the sample is incubated with a capture reagent and a detection reagent containing a detectable label. After sample incubation, a sandwich is formed containing the capture reagent, ADA, and the detection reagent, so that ADA crosslinks the two reagents bound to it, and the bound ADA can be detected. In one embodiment, the immunoassay is a high-throughput assay.

[0077] An ADA crosslinked immunoassay further includes determining the presence or amount of ADA. Therefore, this disclosure provides detection reagents, such as antibodies, AAV vectors, or AAV capsids, conjugated with a detectable label. Non-limiting examples of detectable labels for any of the methods of the present invention include ruthenium, radioactive labels, phosphorescent labels, chemiluminescent labels, fluorescent labels, fluorophores, haptens, electrochemiluminescent labels, or enzyme labels. The detectable labels may be measured using instruments and apparatus known to those skilled in the art.

[0078] This disclosure generally describes assays in which a signal is generated by binding to an ADA drug, such as a viral vector. Assays in which a signal is inhibited or quenched by binding to an ADA drug are also considered. For simplicity, this disclosure considers assays in which a signal is generated by binding to an ADA drug or other reagent, but the methods and compositions described herein may be equally applicable to assays in which a signal is inhibited or quenched by binding to an ADA drug or other reagent.

[0079] As used herein, the term “capture reagent” refers to a chemical substance that can bind an antibody of interest, particularly an anti-drug antibody, and has affinity for a second target in order to capture the antibody of interest. In some embodiments, the capture reagent may be a biotinylated antibody, a biotinylated drug, or a biotinylated virus or viral capsid, which can then be bound to a solid surface via an interaction between biotin and avidin or streptavidin. Other affinity tags suitable for use as capture reagents are known in the art.

[0080] As used herein, the term “detection reagent” refers to a chemical entity characterized by a detectable label capable of conjugating an antibody of interest, particularly an anti-drug antibody, and making the antibody of interest detectable for analysis. In some embodiments, the detection reagent may be an antibody, a virus, or a viral capsid conjugated to HRP or ruthenium. Other detectable labels suitable for use with detection reagents are known in the art and are further described herein.

[0081] As used herein, the term “label” modifies any entity or reagent to include any entity that is conjugated with another empirically detectable molecule or chemical entity (a detectable label). Suitable chemical species for labels include, but are not limited to, ruthenium, radiolabels, phosphorescent labels, chemiluminescent labels, fluorescent labels, electrochemiluminescent labels, affinity labels, quantum dots, or optical dye labels. Labels may also include, for example, biotin, protein A, protein G, or glutathione S-transferase (GST). These labels may be used to label capture reagents or detection reagents.

[0082] Typical fluorophores for use in the methods provided herein include, for example, green fluorescent protein, blue fluorescent protein, red fluorescent protein, fluorescein, fluorescein 5-isothiocyanate (FITC), and cyanine dyes (Cy3, Cy3.5, Cy5, Cy5).5. Cy7), Bodipy dye (Invitrogen) and / or Alexa Fluorine dye (Invitrogen), dansyl, dansyl chloride (DNS-C1), 5-(iodoacetamida)fluorescein (5-IAF), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenzo-2-oxa-1,3-diazole-4-yl chloride (NBD-Cl), ethidium bromide, Lucifer Yellow, rhodamine dye (5-carboxyrhodamine 6G hydrochloride, lysamine rhodamine B sulfonyl chloride, rhodamine-B-isothiocyanate (RITC (rhodamine-B-isothiocyanate), rhodamine 800), tetramethylrhodamine 5-(and 6-)isothiocyanate (TRITC)), Texas Red, sulfonyl chloride, naphthalamine sulfonic acid (including 1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-(p-toluidinyl)naphthalene-e -2-sulfonic acid (TNS), including but not limited to these, anthroyl fatty acids, DPH, parinaric acid, TMA-DPH, fluorenyl fatty acids, fluorescein-phosphatidylethanolamine, Texas red phosphatidylethanolamine, pyrenyl-phosphatidylcholine, fluorenyl-phosphotidylcholine, merocyanine 540, naphthyl styryl, 3,3'-dipropylthiadiadicarbocyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 rhodoacetamide, Cy-5-N-hydroxysuccinimide, Cy-7-isothiocyanate, IR-125, thiazole orange, azure B, Nile blue, Al phthalocyanine, oxacin 1, 4',6-diamidino-2-phenylindole (DAPI), Hoechst Examples include 33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2, calcium green, carboxy SNARF-6, BAPTA, coumarin, phytofluene, coronene, and metal ligand complexes.

[0083] Examples of haptens for use in the methods provided herein include digoxigenin and biotin.

[0084] Enzymes for use in the methods provided herein include, for example, alkaline phosphatase (AP), β-galactosidase, horseradish peroxidase (HRP), soybean peroxidase (SBP), urease, β-lactamase, and glucose oxidase.

[0085] The efficiency and degree of labeling of capture or detection reagents may be influenced based on the relative molar concentrations of the label or tag and the target reagent. Labels may be functionalized, for example, by linking to N-hydroxysuccinimide ester to promote lysine conjugation. In some embodiments, the molar ratio of the label, e.g., biotin or ruthenium linked to N-hydroxysuccinimide ester to the target reagent, e.g., antibody, viral capsid, or viral vector, can range from about 50:1 to about 20,000:1, about 100:1 to about 15,000:1, about 100:1 to about 10,000:1, about 1,000:1 to about 10,000:1, about 9,000:1 to about 11,000:1, greater than about 100:1, greater than 200:1, greater than 500:1, greater than 1,000:1, greater than 2,000:1, It can be greater than 5,000:1, greater than 9,000:1, greater than 10,000:1, approximately 100:1, approximately 200:1, approximately 500:1, approximately 1,000:1, approximately 2,000:1, approximately 3,000:1, approximately 4,000:1, approximately 5,000:1, approximately 6,000:1, approximately 7,000:1, approximately 8,000:1, approximately 9,000:1, approximately 9,500:1, approximately 10,000:1, approximately 10,500:1, approximately 11,000:1, approximately 12,000:1, approximately 13,000:1, approximately 14,000:1, approximately 15,000:1, or approximately 20,000:1.

[0086] In one embodiment, a capture reagent, such as an AAV vector or AAV capsid, is conjugated to a solid surface. In one embodiment, the conjugation of the capture reagent to the solid surface is carried out via a specific binding pair, and the capture reagent is labeled or conjugated. In one embodiment, the specific binding pair (first component / second component) is selected from streptavidin, or avidin / biotin, biotin / neutraavidin, biotin / captavidin, antibody / antigen (see, e.g., Hermanson, GT, et al., Bioconjugate Techniques, Academic Press, 1996), epitope / antibody, protein A / immunoglobulin, protein G / immunoglobulin, protein L / immunoglobulin, GST / glutathione, His-tag / nickel, FLAG / M1 antibody, maltose-binding protein / maltose, calmodulin-binding protein / calmodulin, enzyme / enzyme substrate, lectin / polysaccharide, steroid / steroid-binding protein, hormone / hormone receptor, and receptor-ligand binding pairs. In one embodiment, the capture reagent is conjugated to biotin (as the first component of the specific binding pair). In this case, conjugation to the solid phase is carried out via fixed avidin or streptavidin.

[0087] In one embodiment, the sample tested by the ADA immunoassay is a serum sample. In one embodiment, the serum is human serum.

[0088] In one embodiment, incubation of the sample, capture reagent, and detection reagent is carried out at room temperature. In one embodiment, the incubation time for the sample, capture reagent, and detection reagent is at least 0.5 hours. In another embodiment, the incubation time is at least 1 hour. In one embodiment, the incubation time is at least 1.5 hours. In one embodiment, the incubation time is up to 2 hours. In yet another embodiment, the incubation time is between 0.5 and 12 hours. In one embodiment, the incubation time is between 0.5 and 5 hours. In another embodiment, the incubation time is between 1 and 12 hours. In one embodiment, the incubation time is between 1 and 5 hours. In another embodiment, the incubation time is between 5 and 12 hours.

[0089] In one embodiment, after incubation of the sample, capture reagent, and detection reagent, the sample is transferred to a labeled solid surface, for example, a streptavidin-labeled solid surface, and further incubated so that the capture reagent can bind to the solid surface. In one embodiment, incubation is at room temperature. In another embodiment, after incubation, the sample is analyzed for binding to ADA using any method known in the art for the detection of a detectable label, for example, ruthenium. The true positive signal in the ADA crosslinking assay arises from the divalent binding of ADA to the capture reagent and detection reagent, forming a crosslink.

[0090] As used herein, the term "solid surface" refers to a non-fluid material and includes particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic and ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, and polymer gels; capillaries, zeolites, and other porous materials that may be made from polymers, metals, glass, and / or ceramics; electrodes, microtiter plates, solid strips, cuvettes, tubes, or other containers. The solid surfaces of immunoassays described herein are widely described in the art (see, for example, Butler, JE, Methods 22 (2000) 4-23 (incorporated herein by reference)). Solid surface components of an assay are distinguished from inert solid surfaces that the assay may come into contact with, in that the "solid surface" contains at least one portion on its surface that is intended to interact with the capture agent. A solid surface may be a fixed component such as a tube, strip, cuvette, or microtiter plate, or it may be an unfixed component such as beads and microparticles. Microparticles can also be used as a solid phase for homogeneous surface morphology. Various microparticles can be used to enable either non-covalent or covalent bonding of proteins and other substances. Such particles include polymer particles such as polystyrene and poly(methyl methacrylate), gold particles such as gold nanoparticles and gold colloids, and ceramic particles such as silica, glass, and metal oxide particles. See, for example, Martin, CR, et al., Analytical Chemistry-News & Features 70 (1998) 322A-327A (incorporated herein by reference).

[0091] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, which are either ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA, or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine bases or pyrimidine bases, or other natural, chemical, or biochemically modified, unnatural, or derivatized nucleotide bases. The nucleic acid backbone may contain sugars and phosphate groups (typically found in RNA or DNA), or modified or substituted sugars or phosphate groups.

[0092] Alternatively, the nucleic acid backbone may contain polymers of synthetic subunits such as phosphoramidites, and therefore may be oligodeoxynucleoside phosphoramidites (P-NH2) or mixed phosphoramidite-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing a complementary strand and annealing the strands under appropriate conditions, or by synthesizing a novel complementary strand using DNA polymerase with appropriate primers.

[0093] Adeno-associated virus, or AAV, is a non-enveloped, non-pathogenic parvovirus with single-stranded DNA, a genome of approximately 4.7 kb, and an icosahedral structure. AAV was first discovered in 1965 as a contaminant in adenovirus preparations. AAV belongs to the genus Dependvirus and family Parvoviridae and requires helper function from either a herpesvirus or adenovirus for replication. In the absence of a helper virus, AAV can establish latent status by integrating into human chromosome 19 at position 19q13.4. The AAV genome consists of two open reading frames (ORFs), one for each of the two AAV genes, Rep and Cap. The AAV DNA ends have a 145 bp inverted terminal repeat (ITR) sequence, with 125 terminal bases forming a palindrome, leading to a characteristic T-shaped hairpin structure.

[0094] The Rep gene is transcribed from promoters p5 and p19 into four Rep proteins (Rep78, Rep68, Rep52, and Rep40) that play crucial roles in the viral lifecycle. Proteins Rep78 and Rep68 are encoded by mRNA transcribed from promoter p5. These proteins are essential for regulating viral DNA replication, transcription, and site-directed integration. Two smaller proteins, Rep52 and Rep40, are produced by mRNA transcribed from promoter p19. These proteins are involved in the formation of the single-stranded viral genome for packaging and viral integration. The Cap gene encodes three viral capsid proteins: VP1 (735 amino acids, approximately 90 kDa), VP2 (598 amino acids, approximately 72 kDa), and VP3 (533 amino acids, approximately 60 kDa), which form a viral capsid of 60 subunits in a 1:1:10 ratio. The three capsid proteins are translated from mRNA transcribed from promoter p40.

[0095] Terms such as "envelope," "non-envelope," and "unenveloped" refer to the viral envelope, or the lipid bilayer surrounding the capsid of some viruses. The methods and compositions of this disclosure are not limited to enveloped or non-enveloped viruses.

[0096] A "recombinant AAV vector (rAAV vector)" refers to an adeno-associated virus vector containing one or more heterologous sequences (e.g., nucleic acid sequences not of AAV origin) that may be adjacent to at least one, for example, two AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infecting viral particles when present in host cells that are infected with a suitable helper virus (or express a suitable helper function) and express the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0097] The "capsid" is the protein shell of a virus that encapsulates its genetic material. Three viral capsid proteins, VP1, VP1, and VP3, form a 60-subunit viral icosahedral capsid in a ratio of 1:1:10. A complete capsid contains genetic material and is necessary to provide therapeutic benefits. An empty capsid lacks a genome and therefore lacks the ability to provide therapeutic benefits to the patient.

[0098] A “viral particle” refers to a viral particle consisting of at least one viral capsid protein and a capsid-encapsulated viral genome. While AAV is described as a model virus or viral particle for the purposes of this disclosure, the methods of this disclosure are intended to be applicable to profiling various viruses, such as viridae, subfamilies, and genera. In some embodiments, the viral capsid, virus, or viral particle belongs to a viridae selected from the group consisting of adenoviridae, parvoviridae, retroviridae, baculoviridae, and herpesviridae. In some embodiments, the viral capsid, virus, or viral particle is an atadenovirus, aviadenovirus, ictadenovirus, mastadenovirus, siadenovirus, ambidensovirus, brevidensovirus, hepandensovirus, iteradensovirus, penstildensovirus, amdoparvovirus, abeparvovirus, bocaparvovirus, copiparvovirus, dependoparvovirus, erythroparvovirus, protoparvovirus, tetraparvovirus, alpha-retrovirus, beta It belongs to the genus Virus, selected from the group consisting of retroviruses, delta-retroviruses, epsilon-retroviruses, gamma-retroviruses, lentiviruses, supumaviruses, alpha-baculoviruses, beta-baculoviruses, delta-baculoviruses, gamma-baculoviruses, iltoviruses, Maldiviruses, simplexviruses, variceroviruses, cytomegaloviruses, muromegaloviruses, provosiviruses, roseroviruses, lymphocryptoviruses, macaviruses, percaviruses, and radinoviruses.

[0099] For the purpose of detecting, quantifying, and / or characterizing antibodies against viral vectors, it should be understood that antibodies that bind to viral vectors typically bind to the capsid of the viral vector. Therefore, descriptions of antibodies that bind to viral capsids, viral vectors, viruses, viral particles, etc., should generally be understood as describing the same antibody-capsid binding, and thus assays using viral capsids, viral vectors, viruses, viral particles, etc., as antibody capture and / or detection reagents can be expected to identify, characterize, or quantify substantially the same antibodies.

[0100] "Heterogeneous" means that an entity originates from an entity that is genotypeically distinct from the remaining entity being compared, introduced, or incorporated. For example, nucleic acids introduced into a different cell type by genetic engineering techniques are heterogeneous nucleic acids (which, when expressed, may encode heterogeneous polypeptides). Similarly, a cellular sequence incorporated into a viral vector (e.g., a gene or a portion thereof) is a heterogeneous nucleotide sequence relative to the vector. Heterogeneous nucleic acids that encode polypeptides may be called transgenes.

[0101] An "inverted terminal repeat" or "ITR" sequence is a relatively short sequence found at the ends of a viral genome in opposite orientations. An "AAV inverted terminal repeat (ITR)" sequence is a 145-nucleotide sequence present at both ends of a single-stranded AAV genome.

[0102] As used herein, the term “vector” refers to a recombinant plasmid or virus (“viral vector”) containing nucleic acid that is delivered into a host cell, either in vitro or in vivo. Vectors derived from AAV are particularly attractive for the delivery of genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle fibers and neurons; (ii) they lack viral structural genes, thereby eliminating the innate host cell response to viral infection, e.g., the interferon-mediated response; (iii) wild-type AAV has never been associated with any pathology in humans; (iv) in contrast to wild-type AAV, they can integrate into the host cell genome, and replication-deficient AAV vectors generally persist as episomes, thus limiting the risk of insertional mutagenesis or oncogene activation; and (v) in contrast to other vector systems, AAV vectors do not induce a significant immune response (see ii), and therefore confer long-term expression of therapeutic transgenes (provided their gene products are not rejected).

[0103] A "recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin).

[0104] In some embodiments, the sample is a biological sample. As used herein, the term “biological sample” refers to a sample taken from a living organism, such as a human or a non-human mammal. A biological sample may include, or consist of, whole blood, plasma, serum, saliva, tears, semen, cheek tissue, organ tissue, urine, feces, skin, or hair. A sample may be taken from a patient, for example, a clinical sample. In some exemplary embodiments, a sample may be taken from a non-human animal, for example, a preclinical sample. In some embodiments, a sample is a further processed form of any of the above-described examples of a sample. A sample may include a drug of interest, such as a viral vector, an AAV vector, or an antibody specifically against its capsid. The antibody may be an antibody produced by the immune system of a patient or a non-human animal, or it may be a recombinant antibody.

[0105] As used herein, the term “liquid chromatography” refers to a process in which a biological / chemical mixture carried by a liquid can be separated into its constituent components as a result of the differential distribution of those components as it flows through (or flows into) a fixed liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid (RP) chromatography, ion exchange (IEX) chromatography, size exclusion chromatography (SEC), affinity chromatography, hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), or mixed-mode chromatography (MMC).

[0106] In some embodiments, the methods of the present invention involve the use of size exclusion chromatography. Size exclusion chromatography or gel filtration relies on the separation of components as a function of their molecular size. Separation depends on the amount of time a substance spends in a porous stationary phase compared to the time it spends in a fluid. The probability of a molecule being present in a pore depends on the size of the molecule and the pore. Furthermore, the ability of a substance to penetrate into a pore is determined by the diffusion mobility of macromolecules, which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all, while for very small macromolecules, the possibility of penetration is close to single. Components with larger molecular sizes pass through the stationary phase more quickly, while components with smaller molecular sizes have longer path lengths through the pores of the stationary phase and are therefore retained in the stationary phase longer.

[0107] The chromatography material may include a size exclusion material, which is a resin or a membrane. The matrix used for size exclusion is preferably an inert gel medium, which may be a crosslinked polysaccharide, for example, a complex of crosslinked agarose and / or dextran in the form of spherical beads. The degree of crosslinking determines the size of the pores present in the swollen gel beads. Molecules larger than a certain size do not enter the gel beads and therefore move fastest through the chromatography bed. Smaller molecules such as surfactants, proteins, and DNA that enter the gel beads are delayed in their passage through the bed to varying degrees, depending on their size and shape. Thus, molecules are generally eluted in an order that reduces their molecular size.

[0108] Porous chromatography resins suitable for viral size exclusion chromatography can be made from dextrose, agarose, polyacrylamide, or silica, each possessing different physical properties. Polymer combinations can also be used. The most commonly used is the "SEPHADEX" brand, commercially available from Amersham Biosciences. Other size exclusion supports from different structural materials are also suitable, such as Toyopearl 55F (polymethacrylate, Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, Calif.).

[0109] In some embodiments, the mobile phase used to obtain the eluate from size exclusion chromatography may contain a volatile salt. In some specific embodiments, the mobile phase may contain ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0110] It is understood that the present invention is not limited to any of the aforementioned proteins, antibodies, ADA assays, capture reagents, detection reagents, labels, samples, serotypes, vectors, or liquid chromatography systems, but can be selected by any suitable means.

[0111] The present invention will be better understood by referring to the following embodiments. However, they should not be construed as limiting the scope of the invention. [Examples]

[0112] Example 1. Development of biotin-labeled AAV As described above, there is a need for methods to label and characterize AAVs, for example, for characterizing ADA against therapeutic AAVs. As a first step in developing a diagnostic assay for therapeutic AAVs, we evaluated methods for labeling AAV molecules using biotin and ruthenium. We investigated a lysine-directed conjugation approach.

[0113] The stability of AAV in buffers suitable for lysine-directed conjugations was investigated. AAV1 and AAV8 were stored in various buffers up to pH 8.0, including water, NaH2PO4, KH2PO4, and NaHCO3, and their stability was measured. In particular, the polydispersity index (PDI), a measure of sample size heterogeneity, was monitored. PDI values ​​of less than 20% were considered acceptable. Both serotypes of AAV were found to be stable in buffers tested with 20 mM buffer, as shown in Figures 5A and 5B. No effect on PDI was found when various ionic strengths (additional NaCl or KCl at 0, 20, or 200 mM) were tested.

[0114] Therefore, lysine-directed conjugations with biotin were tested. Lysine-directed conjugations involve incubation of proteins with N-hydroxysuccinimide (NHS) esters, which allows for crosslinking with primary amines (primarily lysine). NHS esters can be linked to tags (or "labels"), such as biotin or ruthenium, thereby covalently bonding the tags to lysine.

[0115] The challenges of AAV conjugation include the limited amount of AAV material at low concentrations, the possibility of aggregation and / or low recovery during concentration and purification, and the limited available information for AAV conjugation. This necessitated an empirical and inventive approach to successfully produce effectively labeled AAV.

[0116] The parameters investigated for AAV conjugates included the ratio of tag molecules to protein moles in the mixture (challenge ratio), the number of available lysines, reaction conditions (e.g., pH and incubation time), the NHS ester solvent, the AAV buffer formulation, and whether the AAV was complete or empty.

[0117] An exemplary procedure for conjugating AAV with biotin is shown in Figure 6A, and includes buffer exchange to basic pH, a challenge ratio of 100–10000x, and purification of labeled AAV. Following successful conjugation, biotin-conjugated AAV8 was successfully purified and characterized using SEC, as shown in Figure 6B.

[0118] Biotinylated AAV was used as a model for anti-drug antibodies, as shown in Figure 7A, and subjected anti-AAV8 mAB mice to a direct assay format anti-drug antibody (ADA) assay. Biotin-labeled AAV was used as the capture reagent, and HRP-labeled anti-mouse IgG was used as the detection reagent. Higher concentrations of biotinylated AAV produced a good signal-to-noise ratio in the ADA assay, as shown in Figure 7B. The direct assay format produced a more nonspecific signal when used to evaluate normal human serum, as shown in Figure 7C. Therefore, we pursued a conventional assay format using labeled AAV, i.e., a conventional assay format using a labeled drug as the detection reagent, to produce a measurable signal.

[0119] Example 2. Development of ruthenium-labeled AAV and determination of DOL Ruthenium-labeled AAV8 was generated using a high challenge ratio and the general process described above for biotin conjugates. MSD® TAG-NHS ester (ruthenium(II) tris-bipyridine, N-hydroxysuccinimide) from Meso Scale Discovery was used as the ruthenium-labeled conjugate. The concentration and degree of labeling (DOL) of the labeled AAV were determined using size exclusion chromatography (SEC), as shown in Figure 8. Protein absorption was measured at a wavelength of 280 nm, and tag absorption was measured at a wavelength of 450 nm. The maximum absorption of the ruthenium tag was approximately 455 nm.

[0120] An exemplary method for calculating DOL is shown in Figure 9A. To calculate the concentrations of AAV and ruthenium, the area under the curve of the main AAV peak was integrated and compared with the standard curve. The ruthenium concentration relative to the AAV concentration was used as DOL.

[0121] The standard curve was generated by analyzing ruthenium at various concentrations using SEC with absorbance measured at 450 nm, integrating the area under the curve (peak area), and deriving an equation relating the peak area to known concentrations, as shown in Figure 9C.

[0122] A comparison of exemplary ruthenium labeling of mAbs and AAVs is shown in Figure 9D. A significantly higher challenge ratio was required for successful labeling of AAVs compared to mAbs.

[0123] We optimized a method for purifying labeled AAV samples, for example, a method for removing unbound ruthenium. AAV8(2.57×10⁻⁶) 13Following a labeling reaction between vg / mL) and MSD® TAG-NHS ester, labeled AAV8 was optionally subjected to a buffer exchange step at a challenge ratio of 1,000:1 or 10,000:1. The recovery of AAV and free ruthenium was then evaluated using SEC, and absorbance was measured at 280 nm and 450 nm. Two buffer exchange systems, the Big Tuna buffer exchange platform (Unchained Labs) and Zeba® Spin Desalting Columns (Thermo Scientific), were compared at a 40K MWCO (molecular weight cutoff). The Big Tuna platform was found to have high efficiency in removing free ruthenium, as shown in Figures 10A and 10B.

[0124] Example 3. Development of an ADA crosslinking assay for therapeutic AAV. As described above, biotin-labeled and ruthenium-labeled AAV8 were produced and used in the ADA crosslinking assay with a mouse anti-AAV8 antibody as a model for ADA, as shown in Figure 4B. High sensitivity was achieved in the ADA crosslinking assay at a higher challenge ratio, as shown in Figure 11A, and at a higher AAV concentration, as shown in Figure 11B.

[0125] A novel ADA crosslinking assay for AAV was tested using human serum. Anti-AAV8 ADA was detected in normal human serum samples at a 1:10 dilution, as shown in Figure 12.

[0126] In conclusion, a wide range of biochemical and biophysical methods were developed to characterize AAV. AAV was successfully labeled with biotin and demonstrated to act in direct ADA binding assays. AAV was also successfully labeled with ruthenium, and DOL was determined using SEC analysis. Compared to prior art in the present art for labeling therapeutic antibodies, labeling AAV required a novel approach, including a challenge ratio of over 1,000:1, to produce labeled AAV that was effective in assay development.

[0127] Using biotin and ruthenium-labeled AAV, we developed a susceptibility and selective ADA crosslinking assay that was able to detect ADA in normal human serum diluted 1:10. These findings provide a basis for diagnostic assays to support methods for detecting labeled AAV and for evaluating immunogenicity in patient exclusion criteria, for example, to support the clinical development of AAV programs.

Claims

1. A method for detecting and / or quantifying antibodies against a viral capsid of interest in a sample, (a) Forming a detection mixture by contacting a sample with a capture reagent, a detection reagent, and a solid surface, wherein the capture reagent and the detection reagent can bind to an antibody against the viral capsid of interest, and the capture reagent can bind to the solid surface. (b) Measuring the detection reagent to detect and / or quantify the antibody against the viral capsid of interest in the sample. Methods that include...

2. The method according to claim 1, wherein the sample is serum.

3. The method according to claim 2, wherein the serum is human serum.

4. The method according to claim 1, wherein the capture reagent comprises biotin.

5. The method according to claim 1, wherein the capture reagent comprises the viral capsid of interest.

6. The method according to claim 1, wherein the detection reagent includes radiolabeling, phosphorescent labeling, chemiluminescence labeling, fluorescent labeling, fluorophores, haptens, electrochemiluminescence labeling, or enzyme labeling.

7. The method according to claim 6, wherein the detection reagent comprises ruthenium or horseradish peroxidase.

8. The method according to claim 1, wherein the detection reagent comprises an antibody.

9. The method according to claim 1, wherein the detection reagent comprises the viral capsid of interest.

10. The method according to claim 1, wherein the solid surface is selected from the group consisting of microplates, resin, agarose beads, and magnetic beads.

11. The method according to claim 1, wherein the solid surface is coated with avidin or streptavidin.

12. The method according to claim 1, wherein the antibody against the viral capsid of interest is an anti-drug antibody.

13. The method according to claim 1, wherein the viral capsid of interest is an AAV capsid.

14. The method according to claim 13, wherein the serotype of the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

15. The method according to claim 13, wherein the AAV is a viral vector.

16. The method according to claim 15, wherein the AAV is a therapeutic viral vector.

17. The method according to claim 1, wherein the viral capsid is a non-enveloped viral capsid.

18. A method for detecting anti-drug antibodies against AAV vectors, (a) Forming a detection mixture by bringing serum into contact with a capture reagent, a detection reagent, and a solid surface coated with avidin or streptavidin, (b) The detection mixture is subjected to an electric current to generate a signal, (c) Measuring the signal and detecting an anti-drug antibody against the AAV vector. Includes, The capture reagent comprises the AAV vector conjugated with biotin, and the detection reagent comprises the AAV vector conjugated with ruthenium. method.

19. The method according to claim 18, wherein the serum is human serum.

20. The method according to claim 18, wherein the solid surface is selected from the group consisting of microplates, resin, agarose beads, and magnetic beads.

21. The method according to claim 18, wherein the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

22. The method according to claim 18, wherein the AAV vector is a therapeutic vector.

23. The method according to claim 18, wherein the signal is an electrochemiluminescence signal.

24. A composition for detecting and / or quantifying antibodies against a viral capsid of interest in a sample, (a) Sample and, (b) Capture reagent and, (c) Detection reagent and (d) Solid surface and Includes, The capture reagent and the detection reagent can bind to an antibody against the viral capsid of interest, and the capture reagent can bind to the solid surface. method.

25. The method according to claim 24, wherein the sample is serum.

26. The method according to claim 25, wherein the serum is human serum.

27. The method according to claim 24, wherein the capture reagent comprises biotin.

28. The method according to claim 24, wherein the capture reagent comprises the viral capsid of interest.

29. The method according to claim 24, wherein the detection reagent includes radiolabeling, phosphorescent labeling, chemiluminescence labeling, fluorescent labeling, fluorophores, haptens, electrochemiluminescence labeling, or enzyme labeling.

30. The method according to claim 29, wherein the detection reagent comprises ruthenium or horseradish peroxidase.

31. The method according to claim 24, wherein the detection reagent comprises an antibody.

32. The method according to claim 24, wherein the detection reagent comprises the viral capsid of interest.

33. The method according to claim 24, wherein the solid surface is selected from the group consisting of microplates, resin, agarose beads, and magnetic beads.

34. The method according to claim 24, wherein the solid surface is coated with avidin or streptavidin.

35. The method according to claim 24, wherein the antibody against the viral capsid of interest is an anti-drug antibody.

36. The method according to claim 24, wherein the viral capsid of interest is an AAV capsid.

37. The method according to claim 36, wherein the serotype of the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

38. The method according to claim 36, wherein the AAV is a viral vector.

39. The method according to claim 38, wherein the AAV is a therapeutic viral vector.

40. The method according to claim 24, wherein the viral capsid is a non-enveloped viral capsid.

41. A composition for detecting anti-drug antibodies against AAV vectors, (a) Sample and, (b) AAV vector conjugated to biotin, (c) AAV vector conjugated with ruthenium, (d) A solid surface coated with avidin or streptavidin Includes, The AAV vector conjugated to biotin and the AAV vector conjugated to ruthenium are modified versions of the same AAV vector. composition.

42. The method according to claim 41, wherein the sample is serum.

43. The method according to claim 42, wherein the serum is human serum.

44. The method according to claim 41, wherein the solid surface is selected from the group consisting of microplates, resin, agarose beads, and magnetic beads.

45. The method according to claim 41, wherein the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

46. The method according to claim 41, wherein the AAV vector is a therapeutic vector.

47. A method for generating a labeled viral vector, (a) Exchange the buffer in the sample containing the viral vector to produce a viral vector sample with a basic pH, (b) The viral vector sample is brought into contact with an N-hydroxysuccinimide ester linked to the label at a basic pH to produce a mixed sample containing the labeled viral vector, (c) Purifying the mixed sample to produce a labeled viral vector. Methods that include...

48. The method according to claim 47, wherein the label is selected from the group consisting of radioactive labels, phosphorescent labels, chemiluminescent labels, fluorescent labels, fluorophores, haptens, affinity labels, electrochemiluminescent labels, and enzyme labels.

49. The method according to claim 48, wherein the label is selected from the group consisting of biotin, ruthenium, and horseradish peroxidase.

50. The method according to claim 47, wherein the viral vector is an AAV vector.

51. The method according to claim 50, wherein the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

52. The concentration of the viral vector in the viral vector sample having a basic pH is approximately 10 per 1 mL. １２ ~about 10 １３ The method according to claim 47, wherein the vector genome is used.

53. The method according to claim 47, wherein the molar ratio of the N-hydroxysuccinimide ester linked to the label to the viral vector is about 50:1 to about 20,000:

1.

54. The method according to claim 53, wherein the molar ratio is approximately 10,000:

1.

55. The method according to claim 47, wherein the viral vector is a non-enveloped viral vector.

56. The method according to claim 47, wherein the purification comprises replacing the mixed sample with a buffer to remove any unconjugated labels.

57. A method for determining the degree of labeling of a viral capsid, (a) Determining the concentration of the conjugated label in the labeled viral capsid sample, (b) Determining the concentration of the viral capsid in the labeled viral capsid sample, (c) Divide the concentration of (a) by the concentration of (b) to determine the degree of labeling of the viral capsid. Methods that include...

58. The method according to claim 57, wherein the viral capsid is an AAV capsid.

59. The method according to claim 58, wherein the serotype of the AAV capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV1, AAV12, a combination thereof, or a variant thereof.

60. The method according to claim 57, wherein the label is selected from the group consisting of radioactive labels, phosphorescent labels, chemiluminescent labels, fluorescent labels, fluorophores, haptens, affinity labels, electrochemiluminescent labels, and enzyme labels.

61. The method according to claim 60, wherein the label is ruthenium.

62. Determining the concentration of the conjugated label is (a) The labeled viral capsid sample is subjected to size exclusion chromatography, (b) Measuring the optical absorption at the wavelength corresponding to the absorption maximum of the label, (c) The peak area is determined by integrating the area under the curve corresponding to the main peak of the labeled viral capsid at the wavelengths of (b), (d) The concentration of the conjugated label is determined by comparing the peak area with a standard curve that relates the peak area to the concentration. The method according to claim 57, including the method described in claim 57.

63. The method according to claim 62, wherein the wavelength is approximately 450 nm.

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